Changes in In Vitro protein-synthesizing activity of embryonic fowl liver

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Title:
Changes in In Vitro protein-synthesizing activity of embryonic fowl liver
Physical Description:
viii, 101 leaves. : ill. ; 28 cm.
Language:
English
Creator:
Chu, Mon-Li Hsiung, 1948-
Publication Date:

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Subjects / Keywords:
Proteins -- Synthesis   ( lcsh )
Liver   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 96-100.
Statement of Responsibility:
Mon-li Hsiung Chu.
General Note:
Manuscript copy.
General Note:
Vita.

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Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
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aleph - 001132259
notis - AFM9582
oclc - 20127592
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Full Text









',I J-iw Iv rr." i: "l-SYN'FT, vZING (FVF
OF ENDIRCYON:C LIVER


















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M-,'`--LI HS I C(;K


A P, S[SRrATRC:U: PFS' .TED TC O I C~ADI: C f:h<.Qf! or


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~I~ -TJ~ F!. I J F LTF. 202 T


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AG "Lf:i' NTS


The author wishes to express her deep appreciation and gratitude

to her research director, Professor Melvin Fried, for his guidance and

encoura ~-,.eijt during the execution of this work.

The author also wishes to express her a-Dreciation to her super-

visory committee members, Dr. R. P. Boyce, Dr. R. J. Mans, and Dr. C.

Moscovici, for their suggestions and criticism.

Special thanks are due to Dr. H. M. Jernigan whose helpful

suggestions contributed much to this work anj to Mrs. S. Eoff for

pFi forming amino acid analyses.

A very special thanks is also e i-,L;ed by the author to her

parents, who have made her education possible, and to her husband,

whose understanding and constant encouragement made this work easier.














TABLE OF CONTENTS


Acknowledgements . .

List of Tables . .

List of Figures . ..... .

Abstract . . .

Introduction . . .

Mechanism and Control of Protein Synthesi
Liver Protein Synthesis .
Research Objectives . .

Materials and Methods . .

Materials . .
Biological Preparations .. .
Biochemical Determinations .

Results and Discussion . .

Characteristics of the Cell-Free Protein-
Sr't L.i Derived From Embryo Livers at
Stages of Development .
Distribution and Activity of Free and '
Polysomes . .
Albumin SInth s. . .

Conclusions . . .

Bibliography . . .

Bicjraphical Sketch . .


Page

ii

iv

v

vii

1

1
6
11

13

13
14
22

30


s .












S. ithesizing
VWrious

...ane-Bound
. .
. -
. .
















. .
. .

. .
















Table
1
I


LIST OF T,- LES



Dependence of protein s r.!.- ;is on various components
of the reaction mixture . .


2 Subcellular distribution of Aminoacyl -1 .A
synthetase activity . .

3 A:nino acid requirement of the pyrophosphate-ATP
excha..:. reaction . . .

4 Ribonuclease activity in liver subcel!i l'r
fractions derived from embryos of various
ag&s . . .

5 Rihonucleuse in iibitor activity in cell sp' derived
fr,., en.bryos of various ages. . .

6 Di sr i but ion of fre and n2e brane-bound polysomes
in h g : nri o.f 'iO-day and 1'--dav !ixt f. .

7 :,'i:n rcid inco; : n.p b !'- ;':!. "so: s befr.)re and aff er Triton


8 Ric'!:"c'e~-e ct i; 'ity in free and inr.irA '":-1oo d





1 Al,'"-'n s K'. i by 30y C X g supX.rpF rt., >rrive
i'- -, c O'" V )" < d L


54



,.








. 62




. 77













LIST OF FIGURES

Figure Page

1 Preparation of total and salt-washed polysomes. ... 15

2 Preparation of total cytoplasmic and free
polysomes from postnuclear supernatant . .. 17

3 Preparation of free and membrane-bound polysomes
from postmitochondrial supernatant . .. 19

4 Effect of cell sap concentration on the [ H] lysine
incorporation into protein . .. 33

5 Effect of polysome concentration on the [ H] lysine
incorporation into protein . .. 34

6 Time course of [3H] lysine incorporation into protein 35

7 Amino acid incorporating activity of cell-free systems
derived from 12-day and 19-day embryos ... 36

8 Amino acid incorporation by total polysomes derived
from embryos at various stages of development .. 38

9 Sucrose gradient profiles of total polysomes
derived from various age embryos. .40

10 Free amino acid content of the cell sap derived
from embryos at various stages of development. ... 43

11 Lysine profiles from amino acid analysis. .. 44

12 Amino acid incorporating activity of cell saps
derived from embryos of various ages. . ... 46

13 Cell sap dependence of [3H] lysine incorporation. .... 47

14 Time course of incorporation of [ 3H] lysine from
[3H] lysyl-ti:';' into polypeptide. . .. 49

15 Effect of cell sap concentration on the incorporation
of [3H] lysyl-tRNA into polypeptide . .... .50

16 Lysine incorporation from [1i] lysyl-tR ;A into polypeptide
by cell saps derived from embryos of various ages 51

17 Time course of pi',,phosphate-ATP excrance assay .. 55









18 Effect of cell sap concentration on the pyrophosphate
exchange reaction . . ... 56

19 Aminoacyl-It"A synthetase activity of cell sap
derived from embrus of various ages. . 58

20 Distribution of free and membrane-bound
polysomes in livers at various stan.: of
development . . 64

21 Sucrose gradient profiles of membrane-bound and
free pol. homes prepared from 19-day embryonic
livers without using detergent. . ... 70

22 Time courses of amino acid incorporation of
membrane-bound and free polysomes . ... 72

23 Sucrose gradient profiles of membrane-bound
polysomes after Triton X-100 treatment. . ... 74

24 Sucrose gradient profiles of free polysomes
after Triton X-100 treatment. . 76

25 Polyacrylamide :-1 electrophoresis of cell-free
protein products released from membrane-bound
and free polysomes before sonication. .. ... .. 81

26 Polyacrylamide gel electrophoresis of cell-free
reaction products released from membrane-bound
and free pol,:: ._s by sonication. . 83

27 Polyacrylamide gel electrophoresis of cell-free
protein products of 16-day membrane-bound
polysomes remaining after precipitation with
antiserum against albumin . .. 8

28 SDS-pol.Ac: rylamide gel elect c',-. is of the cell-
free protein products precipitated with antiserum
against albumin . . 5

29 Polyacrylamide '-1 electrophoresis of cell-free protein
products synthesized by 3,,' X g supernatant from
livers at various sta".:, of develop nt . 90









Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philos.:,_:-,'


CH/.NC.E, IN IIJ VIfO P 'TEIN-SYNTHESIZI';3 ACTIVITY
OF E;7RYONIC FC'.L LIVER

By

Mon-Li Hsiung Chu

March, 1975

Chairman: Melvin Fried
Major Department: Biochemistry

At selected times during chick embryo liver development, a

cell-free protein s-,the3izing system composed of polysomes and cell

sap was prepared. The amino acid incorporating activity of cell sap

decreased with increasing developmental age when assayed using

standard polysones. Polysomes prepared from embryos of different

ages were uniformly active in amino acid incorporation when assayed

with standard cell sap. Cell sap aminoacyl-tRNiA synthetase activity

remained constant during develo,.i-:, nt, whereas the activity in trans-

ferring amino acid from aminoacyl-tRNA into polypeptide decreased with

developmental age. Ribonuclease activity in the embryonic liver in-

creased rapidly with age. The decline in amino acid incorporating

activity of the cell sap is ascribed to the lack of a cell sap component

involved in a stage of protein synthesis subsequent to the amino-

acylation of the tRNAs or to increasing ribonuclease activity, or both.

As development progressed, the free poiyso e content of embryonic

liver decreased, while membrane-bound polysome content increased markedly.

Total polysome content remained relatively constant during development.








The increase in membrane-bound polysome content correlates with and

may be responsible for the increase in the secretion of serum

proteins by liver cells during development. Membrane-bound poly-

somes were less active in amino acid incorporation than free poly-

somes derived from embryos of the same age and this activity difference

was more pronounced in older embryos. The activity of free polysomes

remained constant with age. High ribonuclease activity was found in

the membrane-bound polysome preparation. The difference in amino acid

incorporating activity and ribonuclease activity between free and

membrane-bound polysomes was abolished after polysomes were treated

with Triton X-100. Ribonuclease activity present in the membrane,

therefore, probably inhibits the amino acid incorporating activity of

membrane-bound polysomes accumulating upon maturation of the embryo.

Albumin synthesis in the cell-free :,--t':ri was detected by poly-

acrylamide gel electrophoresis and immunoprecipitation. Membrane-

bound polysomes were found to be the major site for albumin synthesis

in er.l,:i:ic liver cells. The percentage of albumin in total protein

synthesized by membrane-bound polysomes derived from embryos of different

ages was relatively constant. Measurement of total protein syntih-K-.is

by the 3,000 X g supernatant of liver homo'j n:te showed that the

percentage of albumin synthesis increased in parallel with develop-

mental age. Correlations between these results and the differentiation

of serum proteins are discussed.


viii












I r NTrolrii T ION


Mechanism and Control of Protein Synthesis


Spectacular progress in our knowledge of the biochemistry of

protein synthesis has occurred in the past two decades. Much of our

present understanding of the mechanism and control of protein synthesis

has been obtained from studies in cell-free systems. The first such

system was prepared from rat liver (Siekevitz and Zamecnik, 1951;

Siekevitz, 1952; Zamecnik and Keller, 1954). Since then, cell-free

protein-synthesizing systems have been prepared from a wide range of

different organisms. A.i:.ing them are the rabbit reticulocyte system,

first studied by Schweet et at. (1958), and the E. colZ system, first

studied by Lamborg and Zamecnik (1960). From studies on these systems,

it is now known that protein synthesis takes place on ribosomes; that

enzymes ain` factors present in the high speed supernatant of cell

horrogenate (cell sap) are required for protein synthesis; that messenger

RNA (mRNA) provides the code for amino acid sequence of each protein;

that riboson;es move along the ;"'''. as protein synthesis proceeds. Since

several ribosomes can be accommodated on a single "..PUr. molecule, poly-

ribosomes (poil.-oies) ;re formed. The presence of polysomes indicates

that cells are activ-ely :'ynthesizing proteins. The events involved in

protein syrnthesis may be divided into four stages: activation of amino

acids, po! .ptici chain initiation, chain el(. .;tion and chain termi-

nation.









The first step in protein s ..thesis is the ATP-dependent activation

of amino acids by a class of enzymes known as aminoacyl-tRNA synthetases,

each of which is specific for one amino acid (Hoagland, 1955; Hoagland,

Keller and Zamecnik, 1956). Thi activation reaction occurs in two sep-

arate steps with the formation of aminoacyl-adenylate as an intermediate

(Hoagland et aZ., 1957).
M ++
Mg
Amino acid+ ATP+ Synthetase z (aminoacy1 -'P+ Snith -tase) + PPi (1
Mg
(Aminoacyl ;,"', + Synthetase) + tRNA z_ APiin:acyl tRNA+ AMP + Synthetase (2)


Synthetase
Amnino acid+ tRNA < -------- Aminoacyl tRNA+ AMP+ PPi (3)

The activation reaction is usually assayed by measuring the amino acid-

dependent rate at which 3P labeled pyrophosphate is incorporated into

added ATP in Reaction (1) catalyzed by the synthetases (Berg, lE<-).

Initiation of protein synthesis begins with the binding of

initiating aminoacyl-.L':1\ and iHi; to the s -ll subunit of the ribosome

in the presence of GTP and initiation factors to form the "initiation

complex," which then binds to the large subunit of the ribosome to form

a complete ribosome (Haselkorn and Rothman-Denes, 1973). Initiation

factors can extracted from the small ribosome subunit with 0.5 to

2.0 M NH4C1 KC1 (Shafritz et al., 1970; e-v.iood and Thompson, 1971;

Kaempfer and Kaufman, 1972).

Elongation of polypeptide chain can be considered as a three-step

process (Haselkorn and Rothman-Denes, 1973). The first step is the bind-

ing of the incoming aminoacyl-tRNfA to the acceptor site (A site) of the

ribosome. This binding requires GTP and en elongation factor termed

EF-T in prokaryotes and EF-1 in eukaryotes. T- second phase is the









formation of a peptide bond linking the amino .-i',uA' of the aminoacyl-tRNA

with the peptide moiety of the peptidyl-tRNA, located at the peptidyl

site (P site) of the ribosome, to form a new peptidyl-tRNA, one amino

acid unit lcitr :i. This step is catalyzed by the crIy:,mii, peptidyl

transferase, which is an integral part of the large ribosome subunit

(Monro, 1967). The third step is the translocation of the new peptidyl-

tRNA from.the A site to the P site of the ribosome, thus freeing the

A site for the next aminoacyl-tRNA (Traut and Monro, 1964). T!he trans-

location process requires GTP and another elongation factor, termed EF-G

in prokaryotes and EF-2 in eukaryotes.

Polyp,--itide chain termination is signaled by termination codons

(UAA, UAG and UGA) in the mRNA. When a termination codon is reached, the

ribosome binds a release factor, Rl or R2, which activates peptidyl trans-

ferase, which then hydrolyzes the ester linkage between polypeptide and

tF..r, (Caskey et al., 1969; Capecchi and Klein, 1969).

Changes in the protein synthetic capacity of cells have been reported

to occur in response to altered nutritional conditions, hormone stimulation

and various pathological states (Pain and Clemens, 1973). These alterations

of protein synthetic activity are often ascribed to variations in ribosomal

and/or supernatant activities.

Several mechanisms could operate to alter the protein s.rthetic

activity of ribosomes. The rate of protein synthesis depci..;, on the

number of ribosomes per cell, the proportion of ribosomes associated

with ,.:FL.1 and the protein-synthesizing activity of polysomes ('i-,rio

et al., 1953; Wilson and Hoagland, 1967).

Current evidence suggests that chain initiation is the rate

limiting step for translation in most cells. The number of active









ribosomes found in polysomes is r-:;ilated by the initiation process

(Kaempfer, 1971). Messenger-specific initiation factors, capable of

discriminati between classes of mRNAs, have been implicated in the

regulation of protein synthesis in eukaryotes. Heywood (1970)

demonstrated that initiation factors removed from chick muscle ribo-

somes by a high salt wash were required for both the binding of muscle

.-IliA to ribosomes and the mRNA-directed s,..tlhesis of myosin on reticulo-

cyte ribosomes. Ilan and Ilan (1971) reported that, during insect

development, there was a stage-specific initiation factor which pro'; -ed

the formation of the complete initiation complex only with mRNA extracted

from the same stage of development. However, other workers have found no

requirement for specific factors for translation of mRNA in heterologous

system (Lane et ai., 1971; Rhoads et al., 1971; Sampson et al., 1972).

Elongation factors, EF-1 and EF-2, are i'--l:.ent both free in cell

sap and bound to ribosomes. Those factors, when bound to ribosomes, may

influence the activity of ribosomes in a cell-free system. Alexis et a.l:

(1972) found that crude muscle ribosomes from protein-deficient rats showed

lower protein-synthesizing activity than ribosomes from normal rats. The

activity difference between these two kinds of muscle ribosomes were

related to a difference in the content and/or activity of non-ribosomal

factors associated with the ribosome preparations rather than to alter-

ations in the ribosomes themselves. An increase in EF-1 activity in

the spleen of rats was observed following ir.unization (Willis and Starr,

1971). In HeLa cells, EF-2 content increased during rapid growth and

decreased when growth slowed (Hendriksen and Samlson, 1972). In addition,

much of the EF-2 was present in the cell sap .when the growth rate was high,









while a large proportion of EF-2 was associated with 80 S ribosomes

when protein synthesis was restricted.

Aminoacyl-tPNA synthetasesregulate the rate of translation in

several different ways. First, the activity of the synthetases varies

with the rate of general protein synthesis in various physiological states.

Diabetes, which lowers the rate of protein synthesis in rat muscle, also

lowers the activity of synthetases in muscle cell sap (Pain, 1971).

Secondly, changes in the pattern of protein synthesis during growth

and development are often accompanied by changes in the spectrum of

activity of synthetases specific for different amino acids. The

synthesis of phosvitin, a serine-rich egg yolk protein, by the liver

of laying hens was accompanied by an increase in seryl-tP";' synth-:tase

level (Beck, Hentzen and Ebel, 1970). Thirdly, during protein or amino

acid deficiency, synthetase activity rises, facilitating amino acid con-

version into protein. One example of such a phenomenon is that protein

deficiency in the rat results in increased activity of hepatic aminoacyl-

tRNA synthetases (Mariani, Spadoni and To-assi, 1963).

The level of ribonuclease and ribonuclease inhibitor activities

has been shown to vary in different metabolic states of the cell. Kraft

and Shortman (1970) suggested that high inhibitor/ribonuclease ratios

were associated with the state of cytoplas-ic RNA accumulation while

low ratios were associated with net RNA catabolism. The level of

cellular RNA, especially that of mRNA, controls the rate of protein

synthesis. A decrease in ribonuclease activity (Arora and de Lamirande,

1-67) and an increase in ribonuclease inhibitor activity (Shortman,

1962; Moriyama et al., 1969) have been noted in rats following partial

hepatectomy. Protein deficiency in the rat resulted in lower ribo-









nuclease inhibitor activity and higher ribonuclease activity in the

liver cell as compared with well-fed control rats (Sheppard et al.,

1970). The low amino acid-incorporating activity of cell-free systems

derived from chicken liver was ascribed to an uninhibited ribonuclease

in the chicken liver cell sap (Siler and Fried, 1968). Burka (1970)

found a ribonuclease activity in the reticulocyte cell-free system

which was operating during the period when protein synthesis occurred

but was not active at OC.


Liver Protein Synthesis


The liver's special function of secreting considerable amounts of

proteins into blood plasma makes this tissue particularly attractive

to those interested in studying the mechanism of regulation of protein

synthesis. In addition, the liver is a large discrete organ and is

relatively easy to fractionate into subcellular components. Albumin is

the most abundant of all the proteins found in the serum of most

vertebrates, representing 40-50% of the total plasma proteins (Enirle

and Woods, 1960). Some of the early studies on albumin synthesis were

carried out in chicken liver. Peters and Anfinsen (1950) demonstrated

a net synthesis of serum albumin when slices of chicken liver were in-

cubated with 14C02 Peters (1959) studied the intracellular distribution

of the newly synthesized albumin. He found that if the chicken liver

slices were disrupted and fractionated, some albumin was released from

both mitochondrial and microsomal fractions by deox.,cholate treatment.

Of these two fractions, microsomal albumin was found to be more radio-

active. He tested the effectiveness of various reagents in releasing








serum albumin from the microsomal pellet and found that only those

substances which dissolved lipid were effective in releasing serum

albumin. Peters concluded that albumin was held in intimate associ-

ation with the lipid membrane. He also characterized the microsomal

albumin with regard to sedimentation rate, electrophoretic mobility

and N-terminal and C-terminal amino acid residues (Peters et aZ., 1958).

Continuation of such studies in the cell-free system was unsuccessful,

because the microsome fraction isolated fro7 chicken liver was

relatively inactive in amino acid incorporation as compared with that

from rat liver (Campbell and Kernot, 1959). Therefore, the rat liver

has been used as a model system for studying albumin synthesis by most

investigators.

In protein-secreting cells such as liver cells, some polysomes

are bound to the membranes of the endoplasric reticulum and some poly-

somes are free in the cytoplasm (Palade and Siekevitz, 1956). Membrane-

bound polysomes are thought to be Erc-.laged in -aking those proteins

secreted by the cell, whereas free polysones synthesize proteins that

are to be retained within the cell (Siekevitz and Palade, 1960; Vassalli,

1967). Redman (1968) first demonstrated thea: membrane-bound polysomes

were the main or exclusive site of albumin synthesis in rat liver cells.

Takagi and Ogata (1968) made similar findings. Hicks et al. (1969) com-

pared the efficiency of free polysoimes and t tal (free and membrane-bound)

polysomes of rat liver cells in making ferritn, a retained protein,

and albumin in a cell-free system. They si'c.e- that the total polysome

fraction was more efficient for albumin syn-:.esis, presumably because it

contained membrane-bound polysomes. On t-e thier hand, free pol"-iomes









were more efficient in ferritin synthesis. Uiiior,',-,r, and Ono (1972)

reported that albumin was synthesized by free polysomes from 5123

hepatoma, a system in which the synthesized albumin is not secreted

but retained in the cell. Taylor and Schimke (1973) demonstrated

that rat liver polysomal PNA was capable of directing albumin synthesis

in a cell-free system derived from rabbit reticulocytes. The RNA

extracted from membrane-bound and free polysomes of rabbit liver

has been shown to direct albumin and ferritin synthesis in a reticulo-

cyte cell-free system (Shafritz et al., 1973; Shafritz, 1974a).

Albumin synthesis during embryonic development is an interesting

subject for studying the control of protein synthesis. The chick

embryo is an ideal organism for such study, because it is relatively

easy to obtain in large quantities and the ciI'v-,ronic blood is separate

from the maternal circulation. The blood proteins of the chick embryo,

especially in early stages of development, are very different from the

pattern characteristic of adult chicken. There is an increase in the

number of serum proteins as well as an increase in the concentration of

most components with age (Weller and Schechtran, 1962). Albumin first

appears as a distinct component in 9-day serum, its concentration

increasing from 13% of total serum proteins at the 9th day of develop-

ment up to 50% at the 19th day and then showing a slight decrease by

hatching time on the 21st day. The differentiation of serum proteins

from c-i. ,J,'.ic to adult type is probably a direct result of functional

differentiation of the liver cells in their increasing ability to

synthesize adult proteins.

The chick liver appears at the end of the 2nd day of embryonic

life It weighs approximately 2 mg on the 5th day, 50 mg on the 10th








day and 1 g at hatching time (Romanoff, 1967, p. 68). The growth of

the liver, expressed as a percentage of its hatching weight, when

plotted against incubation time, gives a characteristic sigmoid-shaped

curve. However, when the growth rate of liver is plotted against time,

there is a rapid fall from very high to low values between the 7th and

9th day of incubation. Thereafter, the growth rate decreases gradually

until hatching time (Romanoff, 1967, p. 267). It is believed that the

decrease in the specific rate of growth is due to the slowing down of

metabolism caused by the reduction in the rate at which the tissues are

supplied with nutrients (Byerly, 1932). Early enbl;.:l.ic liver cells

lack many ultrastructural features of adult liver cells (Pollak and

Shorey, 1967). Electron micrographs of 5-day embryonic liver cells

show that there is practically no endoplasmic reticulum present in the

cytoplasm. The amount of endoplasmic reticulum increases as develop-

ment progresses, with smooth endoplasmic reticulum appearing prior to

rough endoplasmic reticulum.

There is a considerable amount of information about variations in

enzyme patterns during chick liver development. In 1967, Romanoff

(p. 106) listed 23 enzymes that have been studied in the embryonic

liver. Recently, Greengard and Thorndike (1974) reviewed studies on

52 enr2i..s in the same organ. They classified enzymes according to

the time of their emergence. The majority of enzymes :-Tmer'-i during

embr onic development. Some enzymes attain their mature level at the

earliest time test (about 10th day of incubation), while others are

.il absent at the time of hatching. It is now well established that

each enzyme increases at its own rate, according to its own pattern.








The appearance of new enzymes results in new metabolic potentialities.

However, it is sometimes difficult to interpret the physiological role

of quantitative changes in enzyme levels.

Protein or albumin synthesis in chick E.;-l-ryos has been previously

studied in liver slices or in vivo. Duck-Chong et al. (1964) studied

the protein-synthesizing activity of chicken liver by incubating liver

slices of 8-, 14- and 20-day embryos and adult chicken with [14C]

leucine. They found that embryonic liver slices incorporated [14C]

leucine 15 to 30 times more rapidly than adult liver slices. In the

three different age embryos they studied, the rate of incorporation was

most rapid at 8-day and decreased as development proceeded. Reade et

al. (1965) injected [ C] leucine into 14- to 20-day embryos and then

isolated various proteins from embryonic serum by electrophoresis. They

showed that label was incorporated into the albumin fraction. By

immunochemical techniques, Zaccheo and Grossi (1967) observed that

serum albumin could be detected in the circulating blood of the embryo

at the 4th day of development; however, no albumin was found in the

liver microsomal fraction until the 8th day of incubation. The yolk

sac is the only other embryonic tissue ca.p:hle of producing albumin

(Butler, 1972); albumin thus appears to be supplied to the embryo from

the yolk sac before it is synthesized in the liver.

One approach to understanding the changes involved in the control

of protein synthesis has been the study of a cell-free system. As

previously mentioned, the microsome fraction isolated from chicken

liver was relatively inactive in amino acid incorporation (Campbell

and Kernot, 1l.'). Siler and Fried (15i.) studied the poly (U)-








d.p dcent polyphenylalanine synthesis in both homologous and heter-

ologc'..- systems composed of mixtures of ribosomes and cell sap

prepared from chicken and rat livers and found that with a given

ribosome preparation, chicken liver cell sap was much less active in

ph,,'ylalanine incorporation than rat liver cell sap. This relative

inactivity was ascribed to the presence of an uninhibited nuclease in

chicken liver cell sap. Zimmerman and Fried (1971) found that the

preparation of active ribosomes from adult and embryonic chicken

livers required a higher salt concentration (250 mM KC1) during

isolation than was common in the isolation of ribosomes from rat

liver (50 mM KC1). Jernigan et al. (1972) further studied the effect

of KC1 concentration on the yield and the polyphenylalanine-synthe-

sizing activity of chicken liver ribosomes. KC1 concentrations of

150-250 mM in the isolation medium resulted in higher ribosome yield

and activity than 25-50 :.'1. Polysomal amino acid-incorporating

systems have been prepared from chicken liver by Jernigan ct at.

(1973). The high nuclease activity in chicken liver was minimized by

the use of bentonite and low molecular weight yeast RNA as nuclease

inhibitors. In the presence of such nuclease inhibitors, and under

appropriate ionic conditions, high molecular weight polysomes which

are active in protein synthesis were obtained. Thus the study of the

regulation of serum protein synthesis by components isolated from the

developing chicken liver is feasible.


Research OCjectives

The purpose of this research is to study specific protein

synthesis during chick eiiiu.onric liver development with serum albumin









used as a probe for such study. As a first step in achieving this aim

the following questions were asked. (1) Does the protein synthesizing

activity of the components of a cell-free system derived from the livers

of chick embryos chanir-e during development? (2) Is there a correlation

between the structural differentiation of liver cells with the onset of

serum protein synthesis? (3) Can albumin s',nthesis be measured in the

cell-free system derived from embryonic chick livers? (4) Does albumin

synthesis measured in the cell-free system correlate with albumin

synthesis in vivo?












MATERIALS AND "'TI;ODS


Materials


Animals


Fertile white Leghorn e.g-, were obtained from the Poultry

Science [D :i-rtment of the University of Florida, and incubated, blunt

end up, at 38C in a Leahy model 624 electric incubator. The eggs

were turned three times a day. The age of the embryos was measured

from the start of incubation. Under these conditions of incubation,

the hatching time was 21 days.


Chemicals


Ribonuclease-free sucrose, [ H] lysine, [3H] leucine were purchased

from Schwartz-Mann. ATP, disodium salt; GTP, sodium salt; phosphoenol-

pyruvate, monopotassium salt; pyruvate kinase, Type II from rabbit

skeletal muscle; bovine pancreatic ribonuclease (Type XII-A); Torula

yeast RNA (Type VI); 2,5-diphenyloxazole and 1,4-bis-2(4-methyl-5-
32
phenyloxazole) were purchased from Sigqi Chemical Company. 32P labeled

sodium pyrophosphate and Aquasol scintillation fluid were purchased from

New England Nuclear. Acrylamide and bisacrylamide were purchased from

Eastman Kodak Company. The more common organic or inorganic reagents

were analytical or reagcit grade.








Biological Preparations


Preparation of Total Polysomes


Chick livers from embryos of the desired age were dissected out

and immersed in ice cold Buffer I (150 m M KCI, 8 :.-" MgC!2. 20 mM Tris-HC1,

pH 7.4) containing 250 mM sucrose. The pooled livers were blotted dry

with paper towel, weighed and then i' sized.ize d in 3 volumes of ice-cold

Buffer I containing 27,0 miN sucrose, 5 ..:~/ml bentonite and 78.5 A260 units

(the absorbance at 260 nm)/il yeast RNA at 120 rev./min by 4 strokes of

a glass-Teflon homogenizer. The homo.-rnate .w,'s centrifuged for 15 min

at 12,000 X gmax in a Beckman-Spinco Model L centrifuge to ensure

complete removal of mitochondria (Figure 1). Nineteen parts of this

postmitodcondrial supernatant were mixed with 1 part of 20% Triton

X-100, pH 7.4 and then 3 ml of this suspension were layered over a

discontinuous gradient of 3 ml and 2 ml of Buffer I containing 0.5 M

and 2.0 M sucrose respectively. The tubes containing these gradients

were centrifuged for 2.5 hours at 226,0'0, X gmax in a Spinco "indl L

centrifuge. The pellets were rinsed several times with Buffer I and

then resuspended in 0.25 ml Buffer I/ml original postmitochondrial

supernatant. The suspensions were then clarified by centrifu iution at

12,000 X gmax for 10 min and used immediately for amino acid incorporation

studies. The concentration of soluble polysomes in such preparations was

about 20 A260 units/mi. The A260/-,A ratio of such polysome preparations

was in the r.1'iie 1.77-1.87.








Liver homogenate in Buffer I containing 250 mM sucrose,
5 mg/ml bentonite and 78.5 A260 units/ml yeast RNA


12,000 X gmax, 15 min


Nuclei
Cell debris
Mitochondria


1. Make 1% in Tr
X-100

2. Layer over a
discontinuous
gradient of
Buffer I
containing
0.5 M and
2.0 M sucrose




Resuspend in
Buffer I










12,000



(discard) Tota


Postmitochondrial Supernatant


iton


1. Make 1% in Triton
X-l00 and 0.5 M in K

2. Layer over a
discontinuous gradient
of Buffer II containing
0.5 M and 2.0 M
sucrose


X gmax' 2.5 hr


(discard)


Resuspend in
Buffer II
Layer over 1.0 M
sucrose in Buffer
II


I1
Resuc- pend in
Buffer I


X gmax' 10 min


1


polysomes


(discard)


226,000
2.5 hr


(discard)


Xga
max


(discard)


Salt-wvashed
polysomes


Preparation of total and salt-washed polysomes.


I


ir i


Figure 1.









Preparation of Salt-Washed Polysomes


The postmitochondrial supernatant prepared as described above was

treated with 1/19 volume of 20% Triton X-1C' and made 0.5 M in K+ by

the addition of 2.5 M KC1 (Figure 1). The mixture was then la.er--c

over a discontinuous gradient of 3 ml and 2 ml of Buffer II (500 mM KC1,

8 ML MgCl2, 20 mM Tris-HC1, pH 7.4) containing 0.5 M and 2.0 M sucrose

respectively. After centrifugation at 226,;''. X gax for 2.5 hours, the

pellet was rinsed and resuspended in Buffer II. The suspension was then

lal-.', -J over 2 ml of 1.0 M sucrose in Buffer II, and centrifiu.ul-d again

at 226,000 X gmax for 2.5 hours. The pellet was resuspended in Buffer I

and clarified by centrifugation at 12,000 X g for 10 min. This

suspension is designated "salt-washed polyso-es."


Preparation of Free and M.embrcne-Bound Polvsoes from Postnuclear
Superna tant


The -r_.-paration of free and total cytoplasmic polysomes from post-

nuclear supernatant followed the procedure described by Blobel and Potter

(1967) with some modification (Figure 2). The livers were homogenized in

3 volumes of Buffer I containing 250 mM sucrose, by 15 strokes at 120 rev./

min in a glass-Teflon homogenizer. One milliliter of the 'r.lai2 nate was

mixed with 2 ml of Buffer I containing 2.2 M sucrose. This was then

layered over 1 ml of 2.2 M sucrose in Buffer I. The sample was centri-

fuged in a Spinco SW 50.1 swinging bucket rotor at 170,000 X gax for

40 min. The supernatant was poured off and r;ixed with the material

adheri'',. to the wall of the tube. This vw.s reho7ogenized manually by

4 strokes of a glass-Teflon homo -:izer. The resulting solution was









Liver hu.io'iCioiote in Buffer I containing 250 mM sucrose


Nuclei


Layer
tinuou
Buffer
0.5 M
sucros


150,000
16 hr


over a discon-
s gradient of
I containing
and 2.0 M
e





X gmax


]-------


Free
polysomes


Figure 2.


Make 1.55 M in sucrose

Layer over 1 ml of 2.2 M sucrose in Buffer I

170,000 X gmax' 40 min




Cytoplasm
Cytoplasm


Make 1% in Triton X-100


150,000 X gm
4 hr


Total
cytoplasmic
polysomes


Preparation of total cytoplasmic and free polysomes
from postnuclear supernatant.


T~--P~








designated "postnuclear supernatant." For the preparation of free

ribosomes, 1 ml of postnuclear supernatant was layered over 3 ml of

2.0 M sucrose in Buffer I and the tube was filled with Buffer I con-

taining 250 mM sucrose. The sample was centrifuged in a Spinco Ti

50 rotor at 150,000 X gmax for 16 hours. T,.- pellet contained the free

polysomes. For the preparation of total cytoplasmic polysomes, 1 ml

of the postnuclear supernatant was mixed with 0.4 ml of 20% Triton

X-100. Buffer I was added to it to provide a total volume of 8 ml.

The sample was centrifuged in a Spinco Ti 50 rotor at 150,000 X g
max
for 4 hours. The pellet contained the total cytoplasmic polysomes.

Membrane-bound polysomes were determined by -easuring the difference

between total cytoplasmic and free polysomes.


Preparation of Free and Membrane-Bound Polyso-es from Postmitochondrial
Supernatant


Polysomes were prepared from postmitochondrial supernatant by a

modification of the procedure (Figure 3) described by Blobel and Potter

(1967). The postmitochondrial supernatant prepared as described pre-

viously was layered over a discontinuous gradient containing 2.0 ml and

3.0 ml of 2.0 M sucrose and 1.35 M sucrose respectively in Buffer I. The

gradient was centrifuged at 226,000 X gmax for 4 hours. Free polysomes

sediment in a pellet at the bottom of the tube whereas the membrane-

bound polysomes form a band at the boundary of the two sucrose layers.

The membrane-bound polysomes were removed front the top by suction. Free

polysomes were resuspended in Buffer I by hor-ogenizing by hand. In some

experiments, the free and membrane-bound polysomes prepared under these

conditions were treated with 1/19 volume of 20- Triton X-100'and then












Liver [',i.':i' -n."te in Buffer
5 mg/ml bentonite and 78.5


I containing 250 mM sucrose,
A260 units/ml yeast RNA


12,000 X gmax' 15 min


Nuclei
Cell debris
Mitochondria


Postmitochondrial supernatant


Layer over a discontinuous
gradient of Buffer I con-
taining 1.35 M and 2.0 M
sucrose


226,000 X gmax' 4 hr


1.35 M sucrose---

2.0 M sucrose---


<-Membrane-bound polysomes

---Free polysomes


Figure 3. Preparation of free and rie,:ibrane-bound poly:omes from
postmitochondrial supernat'nt.


I









layered over 2.0 ml of 1.0 M sucrose in Buffer I. The gradients were

centrif'.,- 1 again in a Spinco Ti 50 rotor at 226,i'") X for 2.5

hours.


Preparation of Cel! Sap and Microsomes


One part of liver was homogenized in 3 to 6 parts of Buffer I

containing 250 mM. sucrose at 120 rev./min by 10 strokes of a glass-Teflon

homogenizer (without bentonite and yeast RNA). The hoio .r.nte was

centrifuged at 12, CT X gax for 15 min to prepare a postmitochondrial

supernatant. Cell sap was prepared by centrifuging the postmitochondrial

supernatant at 226,0'1h X ax for 1.5 hours. The resulting microsomal

pellet was resuspended in Buffer I containing 250 mM sucrose.


Prcaration of 3,000 X q Supernatant


Livers were homogenized in 3 volumes of Buffer I containing 250 amM

sucrose at 120 rev./min by 10 strokes of a glass-Teflon homogenizer. The

homogenate was centrifuged at 3,000 X ga for 10 min and the resulting

supernatant was removed froiii the centrifuge tube with a syringe.


Preparation of [3H] lysyl-tFr'


S[3] lysyl-tRNA was prepared from 1-week chick livers by the method

of von Ehrenstein (1967). Fifty grais of livers were homogenized in 3

volumes of Duffer I containing 250 mM sucrose and then centrifuged at

12.0 )C X a for 15 min. The postmitochondrial supernatant was extracted
-Max
two times with an equal volume of buffer saturated phenol. Nucleic acids

were precitated fro;. the aqueous phase by adding 0.1 volume of cold








205 potassium acetate (pH 5) and 2.5 voluv!e of -20C 95% ethanol and

held overnight at -200C. The precipitates ;were centrifuged and

extracted twice with 100 ml cold IM 'I.C1. The supernatants from the

two extractions were combined and tRNAs were precipitated by addi.,.i

2.5 volume of -20C 95% ethanol.

To discharge the amino acids, the i.'..5 were incubated in 20 ml of

0.5 M Tris-HC1 buffer (pH 8.8) for 1 hour at 35C and then reprecipi-

tated by the addition of 0.1 volume of 207 potassium acetate (pH 5)

and 2.5 volume of -200C 95% ethanol.

The tRNAs thus obtained were further purified by dissolving in

10 ml of 0.1 M Tris-HC1 buffer, pH 7.5, and applying the solution to a

DEAE cellulose column equilibrated with the same buffer. After washing

the column with 10 volumes of 0.1 1 Tris-HCl buffer, pH 7.5, tRNAs were

eluted by 1 M NaCi solution in 0.1 M Tris-HCl, pH 7.5, and then pre-

cipitated again by 2.5 volume of -20C 95c ethanol.

To charge the purified tRNAs with [ H] lysine, a final volume of

5 ml contained: 100 mM Tris-HC1 (pH 7.5), 10 rvM M.gC12, 10 mM KC1, 2 mM

ATP, 1 mMl dithioerythritol, 0.5 mCi [3H] lysine (specific activity 10

Ci/mmole), 0.2 mM of 19 other amino acids, cell sap (approximately 3.0

mg of protein from which endogeneous amino acids were removed by passing

through a Sephadex G-25 column), and 10 ig of purified tRNAs. Following

incubation at 350C for 20 min, the mixture was chilled in ice and extracted

with an equal volume of buffer saturated phenol. Charged tRNAs were pre-

cipitated from the aqueous phase as described before. it was then

dissolved in H20 and dialyzed exhaustively against several changes of

H20. The cha,'..J I.,":s were lyophilized and stored at -200C.








Preparation of ':.lease Inhibitors


Bentonite was prepared by a modification of the procedure described

by Fraenkel-Conrat et al. (1961). Fifty grams of bentonitewere suspended

in 1 liter of a solution containing 8 mM MgCl2 and 2 .>I'l EDTA and then

stirred for 3 days. The bentonite was then washed by centrifuging

4 times in 10 mM Tris-HC1, pH 7.4, containing 8 nm1 'jC2, each time

discarding the portion which sedimented in 5 min at 500 rev./min and

the portion which remained in suspension after 10 min at 8,000 rev./min.

The final 500 rev./min supernatant suspension (50 mg/ml) was frozen in

small portions and homogenized before use.

Yeast RNA (Sigma Type VI) was dissolved in H20 by adding sufficient

Tris to maintain the pH at 7.0. The solution was centrifuged at 10,000

rev./min for 10 min, dialyzed at 40C once against 1% EDTA (pH 7.5) and

dialyzed exhaustively against 10 mM Tris-HC1. It was then centrifuged

for 4 hours at 226,000 X gmax and the supernatant was frozen in small

portions. The resulting solution had an A260 nm of 785 units/mil and the

RNA was of sufficiently low molecular weight that no detectable amount

was excluded by Sephadex G-100.


Biochemical Determinations


Amino Acid Incorporation


Incorporation of amino acids into hot trichloroacetic insoluble

material was assayed in a 0.5 ml volume at 35C. Except when noted in

the text, each incubation tube contained: 4.48 A260 units/ml of poly-

somes; 0.1 ml of cell sap (diluted as required with Buffer I); 0.05 ml of








a mixture containing 12.5 pM [ H] lysine or [ H] leucine (125 pCi/ml)

and 10 Jp of each of the other 19 amino acids, 20 mM Tris-HCl (pH 7.4),

150 mM KC1, 0.8 mM ATP, 0.2 mM GTP, 4 mM phosphoenolp.r L.ate, 1 .,-'

dithioerythritol and 20 ug/ml pyruvate kinase (approximately 10 ,.[l

units). All solutions were kept on ice and were added to the incubation

tubes in a shaking water bath just prior to timing. Duplicate or

triplicate 0.1 ml samples were taken from the reaction mixture at

selected time intervals, spotted on 2.3 cm discs of .':itman 3MM paper,

placed in cold 10% (w/v) trichloroacetic acid containing 0.2% unlabeled

lysine (or leucine) for at least 10 min. The paper discs were washed

according to the method originated by Mans and Novelli (1961) and

modified by Bollum (1966), including a 5 min wash in hot 5% trichloroacetic

acid, and then counted in a liquid scintillation counter in 5 ml of

toluene scintillation fluid (4 g/l 2,5-diphenyloxazole and 0.1 g/1

1,4-bis-2(4-methyl-5-phenyloxazolyl)-benzene) (Bray, 1960). The free

amino acids of the cell sap were measured on a Beckman 120C amino acid

analyzer. The specific radioactivity of each incubation mixture was

corrected according to the free amino acid content of each cell sap

sample.


[3H] lysyl-tRNA Incorporation

Cell sap was passed through a DEAE-Sephadex A-25 column prior

to assay. Incubations were in 0.5 ml and contained: 4.48 A260 units/ml

of salt-washed polysomes, 0.1 ml cell sap, 20 mM Tris-HCl (pH 7.4), 6 mM

MgC12, 1 mM dithioerythritol. 150 r KC1, 0.2 mM GTP and 42,000 dpm

[3H] 1;,',,1-tRNA (100 pg tRNA). The reaction mixture was incubated at









350C. At selected intervals, duplicate 0.1 ml samples were spotted

on Whatman I'i filter paper discs which were then placed in ice-cold

10% trichloroacetic acid containing 0.2? unlabeled lysine, washed and

counted as described before.


Aminoacyc "Synthetase Assay


fAinoacyl-tRNA synthetase activity was assayed by the amino acid-
32 32
dependent exchan"_- of P between P labeled pyrophosphate (PPi) and

ATP according to the method of Stulberg and Novelli (1962), with some

modification. The incubation mixture contained the following components

in final volume of 1 ml: 0.1 ml cell sap, 100 mM Tris-HCl (pH 7.4),

5 mM MgC12, 2 iM ATP, 2 mM 32Pi (104-105 dpm), 50 mM KF, 1 mM dithio-

erythritol and 2 mM of each of 20 amino acids. The reaction was

initiated by the addition of cell sap and incubated at 35C for 15

min. The reaction was terminated by the addition of 0.5 ml of 7%

HC104 followed by 0.2 ml of a suspension of Norit A (200 mg dry weight).

Four milliliters of 0.1 M sodium acetate were added to the mixture which

was then mixed -::t.h- :.hly with a wooden applicator stick. The adenosine

phosphates were absorbed by the Norit A and inoc.;3nic phosphates were

left in the solution. Norit A was separated by low-speed centrifugation,

resuspended in 5 ml of 0.5 M acetate buffer plus 0.1 M PPi (pil 4.5),

mixed thoroughly, centrifuged and the supernatant discarded. The wash-

ing procedure was repeated 3 times with 5 ml of 0.05 M acetate, pH 4.5

(no PPi) and once with 5 ml of H20. Tile Norit was then suspended in

5.0 ml of IN HC1 and heated at 100C for 15 Kin to hydrolyze the terminal

phosphate groups of ATP. After hydrolysis, the mixture was centrifuced









and 1 ml aliquots of the supernatant were removed and then counted in

a liquid scintillation counter.


Ribonuclease Assay


The ribonuclease assay is essentially a ;odification of the

procedure of Anfinsen et al.(1954), a method based on the liberation

of acid-soluble oligonucleotides from yeast R;A. The RNA substrate

was purified by the method of Utsunomiya and Roth (1965). Yeast RNA

(Sigma Type VI) was dissolved in H20 and the pH was adjusted to 7.0 by

adding Tris. The solution was dialyzed against 5 changes of 2.5

volumes of 0.025 M EDTA (pH 7.0) and then against 5 changes of 5.0

volumes of H20. The concentration of the dialyzed RNA was adjusted

to 1% as determined by absorbance measurement. The purified RNA thus

obtained was stored frozen until use and this material gave very low

blank value. Ribonuclease activity was measured by incubating, at 350C

for 1 hour, 0.4 ml of 1% purified yeast RNA, 0.1 ml of 200 mM Tris-HCl

(pH 7.4) and 0.5 ml of enzyme preparation. Triplicate samples were

assayed when possible. The reaction was terminated by the addition of

0.5 ml ice-cold 0.75% uranyl acetate in 25c HC104. The mixture was

kept at o0C for at least 15 min. After centrifugation at low speed,

the supernatant was diluted 10 times with H20 and the absorbance at

260 nin was measured in a Beckman DU spectrophotometer against a zero-

time blank (containing both enzyme sample and RIA). Another blank

without enzyme was treated in the same way in order to correct for non-

enzymatic depolymerization of -.rJ\.









Ribonuclease Inhibitor Assay


Ribonuclease inhibitor activity was -easured according to the

method of Roth (1956) with some modification. A final volume of 1.0

ml contained: 0.1 ml of a solution containing 0.0125 pg of pancreatic

ribonuclease, 0.4 ml of 1% purified yeast RA, 0.1 ml of 200 mM

Tris-HC1 (pH 7.4) and 0.4 ml of cell sap. The mixture was incubated

at 350C for 60 min and the reaction was terminated by the addition of

0.5 ml of 0.75'. uranyl acetate in 25% ;:.10). The A260 of the sample

was measured as described previously. Controls containing 0.4 ml of

Buffer I instead of cell sap, and others containing 0.1 ml of H20 in

place of pancreatic ribonuclease were assa -d simultaneously.


Analysis of Cell-Free Reaction Products


For analysis of protein products, a 2-5 ml reaction mixture was

used. Each ml of reaction mixture contained 5-10 A260 units of membrane-

bound or free polysom es, 1 mg cell sap protein, 20 mM Tris-HC1 (pH 7.4),

100 mM KC1, 6 mM MgCl2, 0.8 mM ATP, 0.2 ml' GTP, 4 mM phosphoenolp., ,...ite,

20 pg pyruvate kinase, 1 mM dithioerythritol, 1 pM each of [3H] lysine

and [3H] leucine (12.5 ,iCi per ml reaction mixture), 1 IpM of each of

the other 18 amino acids. Incubation was performed at 35C for 60

min. Subsequent treatment of the sample ..as carried out essentially

by the method of Koga and Tamaoki (1974) At the end of the incubation,

the reaction mixture was centrifuged at 15,03C X gax for 1.5 hours

to sediment polysornes. The supernatant was dialyzed extensively against

several cahiges of 20 Tris- HCl (pH 7.4) and then subjected to poly-








acrylamidi gel electrophoresis. The pellet was resuspended in the

original volume of 20 :u!- Tris-HC1 (pH 7.4). The sample was frozen in

dry ice-acetone, thawed and sonicated for 5 min. This treatment was

repeated three times. The disrupted sample was centrifuged at 150,000

X gax for 1.5 hours and the resulting supernatant was dialyzed as

above and subjected to gel electrophoresis. Duplicate 10 pi samples

were taken from the reaction mixture in each step and spotted on filter

paper discs. Hot trichloroacetic acid insoluble materials were determined

as previously described.

In case of the 3,000 X g supel-rntant system, incubation for

amino acid incorporation was the same as with the polysomal system except

that 0.2 g equivalent of liver per ml reaction mixture was substituted

for polysomes and cell sap. Subsequent analysis of the reaction products

was as described above.


Polyacrylamide Gel Electrophoresis


Gel electrophoresis in the absence of SDS was performed in 7.5%

gels (acrylamide:bisacrylamide, 37.5:1) at pH 8.9 according to the method

of Davis (1964). Between 5,000 to 15,000 cpm of hot trichloroacetic acid

insoluble material was applied to the gels. The current was adjusted to

2 m Amp per tube for the first 20 min, then it was increased to 5 m Amp

per tube. Total running time was 2 hours. After electrophoresis, the

gels were sliced into 2 mm sections which were dissolved by incubating

in 0.2 ml of 30% H202 at 60C in tightly capped scintillation vials and

then counted in 10 ml of Aquasol scintillation fluid.

SDS-polyacrylamide gel electr-i'lh:.resis was performed in 7.5% gel

at pH 7.1 according to the method of Palmiter ct a,. (1971). The current








was .zJiusted to 2 m Amp per tube for the first 20 min, then it was

increased to 5 m Amp per tube. Total running time was 4 hours. Radio-

activity in the gel was determined as described above.


Imunop rec ipiati on


A monos-. -:ific antiserum to chicken serum albumin was produced in

a goat by three alternate weekly intramuscular injections of purified

chicken albumin. Fifty microliters of the antiserum would precipitate

between 6 to 12 pg of albumin at the equivalence point. For immuno-

precipitation of the cell-free reaction p'-nldu'.rs, 10 ig of purified

albumin were mixed with 100-200 il of sample followed by the addition

of 100 il of antialbumin serum. The mixture was incubated at 350C for

60 min and then at 4C for 24 hours. The sample was centrifuged at

low speed for 10 min at 4C. The precipitate was washed three times

with 20 mM Tris-HCl (pH 7.4) in 150 mM NaCl, and dissolved in 1% SDS,

1% 0-mercaptoethanol and 20 mM Tris-l.l (pH 7.4) by boiling for 5 min.

Sucrose to a final concentration of 10% and Bromphenol blue tracking

dye were added to the sample and SDS gel electrophoresis was performed

as described above.

For assay of albumin sr nth:e-is, immunoprecipitation was performed

as described above except in the presence of 1% sodium deoxycholate and

1% Triton X-100 to reduce nonspecific precipitation (Shafritz, 1974b). The

immunoprecipitate was washed three times with 20 mM Tris-HCl (pH 7.4),

150 mM NaC1, 1% sodiurn deo;. :'jDlate and 1% Triton X-100 and then dissolved

in 0.04 N NaIC'. The radioactivity of this solution was determined by

counting in 10 ml of Aquasol scintillation fluid. A blank containing








nonimmune goat serum was treated under the same conditions and its

radioactivity subtracted from the radioactivity in the inmuno-

precipitate.


Sucrose Gradient Centrifugation


Linear sucrose gradients (36 ml, 15-40% sucrose in Buffer I)

were prepared in an ISCO, Model 570, rifadient former. Approximately

2.0 to 5.0 A260 units of polysomes were applied to each gradient and

centrifuged at 131,000 X gmax for 2 hours in a Spinco SW 27 swinging

bucket rotor. The bottom of each tube was punctured and the gradient

was displaced upwards with a 50' sucrose solution. The gradients were

monitored at 254 nm with an ISCO Model UA-4 ultraviolet analyzer.


Measurement of RNA and Protein


RNA concentration was determined by the method of Fleck and Munro

(1962). Protein was assayed by the method of L.:r,' et ca. (1951) using

bovine serum albumin as a standard.


Statistical -Analyses


The results were expressed as mean value + the standard error when

more than three separate experiments were done. Other results were

expressed as the average of duplicate or triplicate determinations

when the values were derived from a single representative experiment.












RESULTS AND DISCUSSION ;


Characteristics of the Cell-Free Protein-S.'tthesizing System
Derived from Enmbryo Livers at V _;_ ; Stages
c r [ ,:. -. I


Requirements of Cell-Free Amino Acid Incorporation


The cell-free amino acid incorporating system used in this

investigation was composed of polysomes and cell sap prepared from

chick embryo livers. Various conditions were tested to achieve

maximal activity, and the method of preparation outlined in Materials

and Methods was found to be most satisfactory. KC1 concentrations of

150-250 mM in the isolation medium resulted in hi'-ier polysome yield

and activity than 25 mM. In this study, 150 r KC1 rather than 250

mM KC1 was used because hi iler KC1 concentration might increase the

risk of ribosome dissociation. Preliminary experiments showed poly-

sones isolated in 150 mM KC1 to be slightly r-ore active than those

isolated in 250 .1' KC1. MgC12 concentrations below 5 mM in the

isolation buffer reduced the yield of polyso.es. The use of more

concentrated sucrose (2.0 M versus 1.5 M) in the lower layer of the

discontinuous gradients eliminated most of the protein impurities.

Polysomes isolated from livers of embryos older than 11 days showed

degradation by nuclease. Addition of bentonite and low molecular

weight yeast ;';4 to the isolation buffer as r~clease inhibitors

protected polysomes from degradation. Bentcr:t or yeast RNA alone









reduced the nuclease activity; however, a combination of the two

proved best.

Incorporation of amino acid into hot trichloacetic acid insoluble

material d,..rin.ded on the presence of polysomes, cell sap and an energy

source (Table 1). Figure 4 shows the effect of increasing amounts of

cell sap on amino acid incorporation by a fixed amount of polysomes.

Figure 5 represents another type of experiment in which the amount of

cell sap was fixed and increasing amounts of polysomes were added.

Within the concentration ranie; tested, incorporation was linearly

dependent upon the amount of both cell sap and polysomes. The system

could not be saturated with cell sap probably because one or more

active components were in low concentration. Use of lower polysome

levels would have reduced the lysine incorporation to less than

significant levels. A typical time course of incorporation is shown

in Figure 6. The incorporation was linear with time for about 10

minutes.


Polysome Activity


The amino acid incorporating activity of cell-free systems derived

from embryos of different ages was compared. Polysomes prepared from

12-day and 19-day embryonic livers were incubated with homologous

cell sap. As shown in Figure 7, the cell-free system derived from

12-day embryos was found to be more active than that from 19-day

embryos. To investigate this difference further, the activity of

polysomes derived from embryos of various ages was compared. For

this purpose, the assays were set up so that the polysome age was the












Table 1

Dependence of Protein Snthesis on Various
Components of the Reaction Mixture


Polysomes and cell sap were prepared from 17-day embryos and
incubated for 15 min at ?TC in the complete reaction mixture
described.in Materials and Methods with 0.1 mg cell sap protein.
Incorporation of [3H] lysine was determined as acid soluble material
assuming 380 cpm = 1 pmol lysine incorporated. The values given are
the means of triplicate reaction mixtures assayed in triplicate.

pmoles of [ H] Lys
System incorpd/mg t F .A


Complete 364.0

Complete less polysomes 10.6

Complete less cell sap 2.8

Cr:,:plete less energy source* 3.4


*ATP, GTP, Phosphoenolpyruvate














































0.4 0.8 1.2 1.6 2.0

Cell Sap (mg protein/mi reaction mixture)


2.4


-3
Effect of cell san concentration on the [3H] lysine incor-
poration into protein. Polysomes and cell sap were prepared
from 19-day emb;,r.s. Incubation was performed at 35C for
15 min as described in Materials and '-trhods with 0.2 mg
ribosomal RNA and indicated level of cell sap protein per
ml reaction mixture. Each point represents the a'.c,.1,, of
duplicate reaction mixtures assayed in triplicate.


Fi -;uie 4.












































































































pdjocul sfI|. [H.] Jo salowd
I


*r- vI *r-
e" E- O
S- 0-

S-> 0L --

0 Uo O
S- U1 ro 4-
CO 0- 0. CL rco
SE u

4-' C: CD

4- -)- 3 S.-
C r .C 4-J 4--
O .C 4- X
0 --
*a 3 *- *r-



4 .-' u)


X u 0 D fl3 m
*r- C r S c


0 C ) E C'- L.
0 C: ) n E :3
o CO OrEC


43 O no QL E
O I Er-

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--- O) 4-' C *i-
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L- E -l-

a: a0)- in

E- r-0 L
C 0 -D 0C 0

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) *- U r-
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i L S (0 0


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0- CL QC C)
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4-' S-
01 -- (I ro 0)

\J O O) r- 0C


r- 4-- .C


0 C V)
4-- v D 1- -'C
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E C U
4- 0 4-' *r- in
U to rc 0) C0
0) > -' S-
4- +4-)' CL
4- O s. 0)
LLJ CL 0 CL 5




L.

0)

5-

l-

























500


z2
s.-
r 400
E



U
o

0)
300



r
'' 200
+-

0

E
- 1ir,-


10 15 20 25 30


Ti me (T-i inf


Fi gure 6. Ti -Ie ce~~.CF'I .



dPlys 1L S( ) ; C.2 ,K -'.ii 'a-p r
filr an'2' dir>.






























































5 10


Time (rin)


Amino acid incorporating activi-y of cell-free systems
derived from 12-day and l '--. es-bryos. Polyso -es pre-
pared from: 12-day or 19-day e::', .'nic livers were incubated
with homologous cell sap (1. -3 cell sap protein per ml
reaction mixture) as described in materialss and Methods.
The incubhtio n was for various ri:es at 350C. The values
given are the means of triplclcte reaction miixtures assayed
in dc..;:licate. o, 12-day e:-:b"cs; o, 19-day eiin ryos.


500





400


20 0


s-

E
o-







o
0




r-




0
Ei

0
E
0.


Firie- 7.








only variable in the reaction mixture. Polysomes from embryos of

different ages were prepared at the same time and incubated in the

presence of a standard cell sap preparation. As can be seen in

Figure 8, no significant differences in polysome activity were observed.

The sucrose gradient profiles of these polysome preparations are shown

in Figure 9. High molecular weight polysomes can be isolated from

embryonic livers throughout the developmental period studied. The

ratio of polysomes (10-30 ml from meniscus) to monomers (6 ml from

meniscus), indicated by the area under the profiles, decreased with

increasing developmental age. The polysome peak was found to be

nearer the meniscus in sucrose density gradients with increasing age,

indicating a decrease in average polysome length with maturity.

Since these polysomes were isolated, resuspended and then analyzed

by sucrose gradients, subtle differences in the profiles may be due

to minor variations in the complex isolation procedure.


Cell Sap Activity


The activity of cell saps derived from embryos of various ages

was also compared using a standard polysome preparation. The con-

centration of free amino acid in each cell sap preparation varied

quite significantly which caused significant .:l,'ngcs in the specific

radioactivity of each reaction mixture. Therefore the free amino acid

concentrations of each cell sap sample were determined in a Beckman

amino acid analyzer and used for corrections in the specific radio-

activities when calculating activities of different cell sap samples.

When such corrections were made, the incorporation of amino acid was



















T `?


50 [-


Embryo age (days)


Figure 8.


Aniino acid incorporation by total polysomes derived from
embr yos at various stages of development. Polysomes were
isolated from five different 'e e-,bryos on the same day.
Each polysome preparation was ic'ubated with cell sap
prepared from 19-day embryos (1.0 -g cell sap protein/mi
reaction mixture) and other cc- rents as described in
Materials and Methods. Incub;.r'cn was for 15 min at 35'C.
Tihe values given are the means + S.E. of three samples of
each age.


250







< 200


E
01
0O
0
= 150

-J
r-"


100
0
o
E
0-


--I _1__ _L --- ----L ~------~-----r~---p----


























Sucrose gradient profiles of total polysomes derived
from various age embryos. Polysomes were prepared as
described in Materials and Methods. Between 2 to 4 A260
units of polysomes were layered on each 15-40% sucrose
gradient. The gradients were centrifuged and monitored
as described in Materials and Methods. (A) 10-day poly-
somes, (B) 12-day polysomes, (C) 15-day polysomes, (D)
17-day polysomes, (E) 19-day polysomes.


Figure 9.
























































10 20 30
ml from meniscus








proportional to cell sap concentration in the r -ie usually assayed.

In all assays of cell sap activity, duplicate determinations at three

different cell sap concentrations were done to assure that comparisons

were made at the linear part of the incorporation versus cell sap

concentration curve.

At the beginning of this study, [ 3H] lysine was used for all

incorporation studies. During the analyses of cell sap samples, two

interesting observations were made. First, the free lysine concentration

in the cell sap appeared to be age-dependent. Lysine concentrations

of 12-13 day and 18-20 day cell sap were always considerably lower

than the other days (Figure 10). In order to investigate whether the

concentrations of other amino acids were also age-d:pendent, complete

amino acid analyses were performed for all cell sap samples. Some

representative results are shown in Figure 10. The concentrations of

leucine, histidine and methionine was constant throughout development,

while those of other amino acids showed variations with age. The

patterns of arginine and lysine were similar in that the variations

in concentration were parallel.

The second observation was that the lysine peak from embryonic

cell sap, as eluted from the amino acid analyzer, was not symmetrical.

As can be seen in Figure 11, it appeared that another component of

cell sap eluted a little earlier than lysine did and formed a shoulder

on the lysine peak. Attempts to identify this other component by

comparing with several possible known compounds ,ere unsuccessful.

Changing the pH of the eluting buffer did not furtlir separate the

two components. It is interesting that the phenc:enon is not observed




























Fi:ure- 10.


Free amino acid content of the cell sap derived from
embryos at various stages of development. Cell sup
proteins were precipitated with 1'-- trichloroacetic
acid and the resulting supernatant was analyzed on a
Beckman 120C amino acid analyzer. The values represent
the average of at least three dceer~r inrtions. The
lysine values were the average of 5 to 7 determinations.
0, lysine; n, valine; argininte; histidine;
^ leucine; O, methionine.















18.0




16.0
C
r-



14.0




~12.0-
E

o
E

S10.01-

@ Lys

Si Val
u 8.0
o
- Arg

6.0

E
^ His

4 O ". .ALeu




2.0


-- --------- --- Met


9 11 13 15 17 19
Embryo age (:'js)




44













-,







I-
e-



C)




cv

-0





I






0 r-


c I r-
L r "
--- O- -



"-(0


I---J





















---


SOF-
r*-
-* ,


















o -












0 r-






Sr-
r --
Et



















LI -M
f-*-- "~" ro L








in cell sap from adult chicken liver. At this point, all that can be

said is that the cell sap from embryonic chick liver apparently contains

a ninhydrin-positive component which is either absent or present in very

low concentration in the cell sap from adult chicken liver.

To ascertain that the measurement of lysine concentration did not

introduce artifacts in the assay of cell sap activity, [3H] leucine

was used in some experiments. Figure 12 shows the cell sap activities

at different stages of development using either [3H] lysine or [3H]

leucine. Cell saps from young embryos were more active in amino acid

incorporation than those from older embryos. Similar results were

obtained with either labeled amino acid. This indicates that correction

of the specific radioactivity by amino acid analysis is reliable.

Figure 13 shows the effect of cell sap concentration on the incorpor-

ating activity of cell sap prepared from two different age er.br.,s.

The age-related activity difference was observed at all concentrations

tested.

It is known that supernatant factors tend to bind to crude poly-

somes and that they can be removed by washing polysomes with 0.5 M KC1.

The factors bound to crude polysomes may introduce a significant back-

ground incorporation when cell sap concentration is limiting and this

background incorporation may interfere with cell sap activity measure-

ments. To test this possibility, cell sap activity was studied using

0.5 M KC1-washed polysomes. The results are shown in Figure 12. In-

corporation was reduced considerably as c,:...;t:.ered with that measured

using crude polysomes, but similar age-related changes were obtained.

The above experiments su --St that cell sap from young embryos

is more active in amino acid incorporation than that from e'!'r.,.'s at





















































~I _


Embryo age (days)


Figure 12.


Amino acid incorporating activity of cell saps derived from
emb,.o: of various ages. Cell sacs prepared from livers of
different age .- ,-.os were incubated with a standard crude or
salt-washed polysome preparation at 3"5C for 15 min as described
in Materials ano Methods. The activity was determined using
three different cell sap conceneations (between 0.5 and 1.5
mg protein per ml reaction mixtu:e). The results have been
normalized to incorporation per -g cell sap protein. Each point
represents the mean + S.E. of tree separate experiments. The
activity was measured with either [3H] lysine or [3H] leucine.
@, [3H] lysine incorporation, crude oolysomes; o, [3H] leucine
incorporation, crude polys. ; [3.] lysine incorporation,
salt-washed polysoi.es: a, [-] "eucine incorporation, salt-
washed polysomes.


500


400


S-

E

o
.r,-


_J
S-





r-




LJ
I-I
0
o







E
M_
l-I


0-


200E




















1000



800



600




400


200


Figure 13.


0.4 0.8 1.2 1.6 2.0 2.4

Cell sap (mg protein/mi reaction mixture)

Cell sap dependence of [3H] lysine incorporation. Polysomes
prepared from 16-day embryonic livers were incubated with
cell sap prepared from 9-day or 16-day e-mbr.ioic livers under
the conditions described in Materials and Methods. Each point
represents the average of duplicate reaction mixtures assayed
in triplicate. o, 9-day cell sap; e, 16-day cell sap.








later staj-:, of development. The results of Duck-Chong et aZ. (1964)

also showed that liver slices from young embryos incorporated amino

acid more rapidly than those from old ei'br',os. Whether the decline

in amino acid incorporation reflects the in vivo protein synthesizing

capacity is not clear. One possible correlation seems to be with the

decrease in ,-,..':th rate of liver during development (Romanoff, 1967, p.

267).


[3H] lysyl-tRNA Incorporation


Cell sap contains Er,.j.-, and other factors which are required

for different steps in protein synthesis. In the following experiment,

the ability of cell sap to transfer labeled amino acid from aminoacyl-
-3
tRNA into polypeptide was tested. [3H] lysyl-tRNA was prepared by

charging chick liver tRNA with [3H] lysine. To avoid differential

dilution of added [ H] lysyl-tRNA with endogenous lysyl-tRNA, the

cell sap was passed thrmi')h a DEAE-Sephadex A-25 column to remove the

endogeneous tRNAs. The extent of incorporation was assayed using a

salt-washed polysome preparation. Under these conditions, the in-

corporation of radioactivity was linear with time for at least 15

minutes and was depni.ent upon the amount of cell sap added (Figures

14 and 15).

The incorporation by cell sap derived from embryos of different

ages is shown in Figure 16. Cell sap from young embryos was more

active in the transfer of amino acid into polypeptide than cell sap

from older embryos. This is consistent with the previous observation

when labeled ar!ino acids were used for incorporation, although the age-

related differences were not exactly parallel.





























E
-U
C'


5 10 15


Time (min)


Figure 14.


Time course of incorporation of [3H] lysine from [3H] lysyl-
tRNA into pol .l -.tide. Salt-washed polyson:es prepared from
20-day embryonic livers were incubated with the cell sap
prepared from 13- or 20-day e., Lb,-.'.,ic livers under the con-
ditions described in Materials and '-.thods. Each ml
reaction mixture contained 1.0 .-j cell sap protein. The
values given are the means of triplicate determinations.
o, 13-day cell sap; o, 20-day cell sap.






















120






100


z
or
L
o,
E
E
n
-cl
x
m
1
o


0.4 0.8 1.2 1.6 2.0


cell sap (mg protein/mi reaction mixture)


Fign-ire 15.


Effect of cell sap concentration on the incorporation of
[3H] lysyl-tRNA into polypepti'e. Polysomes and cell sap
were prepared from 16-day embry\:ic livers. The reaction
conditions have been described ir trialsas and Methods.
Each point represents the average e" duplicate reaction
mixtures assayed in triplicate.













































10 12 14 16 18 20


Embryo age (days)


Figure 16.


Lysine incorporation from [3H] lysyl-tRNA into polypeptide by
cell saps derived from ;L :.os of various ages. Cell saps
prepared from embryos of different ages were incubated with
a standard salt-washed polysome preparation as described in
Materials and ci tods. 7he -incorporation was determined
using thr-e di ferent ce!l sap concentrations (between 0.5
and 1.5 mn protein per ml reaction mixture). The results
have been normalized to incorporation per mg cell sap
protein. Each point represents the mean + S.E. of three
separate .i.'-rinments.








Aminoacy1-tRP\ Synthetase Activity


The ability of cell sap to activate amiro acids was studied.

Aminoacyl-tRNA synthetase activity was assa..e: by the exchange of 32P

labeled p-,i,:hosphate with the pyrophosphoryl moiety of ATP in the

presence of amino acids. There is some evidence that synthetases

are associated with large particles (Hampel and Enger, 1973). The

distribution of synthetase activity in both the cell sap and micro-

somal fraction was studied. As shown in Table 2, while some activity

was found in the microsomal fraction, more than 90' of the synthetase

activity was found in the cell sap. Preliminary experiments were

performed by measuring the synthetase activities of different age

embryos using both postmitochondrial supernatant and cell sap.

Parallel results were obtained. Therefore, in the following assay

cell sap was used as the source of enzyme.

While the pyrophosphate excharn:- reaction is dependent on the

presence of added amino acids (Table 3), there was a high background

exchange between pyropho-l'.-,.ite and ATP in the absencee of added amino

acids. Dialysis of the sample reduced the back-'..iad considerably.

After subtracting the blank without added amino acids, the activity

of the sample before and after dialysis was essentially the same.

The time course of pyrophosphate exchare is shown in Figure 17.

The reaction was linear with time up to 20 minutes. The exchange

reaction is linearly dependent uL'i-'. the cell Sap concentration thus

permitting the comparison of synthetase activity under these assay

conditions (Fi.;',re 18).













Table 2

Subcellular Distribution of Aminoacyl-tRNA
Synthetase Activity


The cell fractions were prepared from 19-day embryonic livers
by h-r, .'i:i.,izing livers in 5 volumes of Buffer I containing 250 mM
sucrose. Postmitochondrial supernatant and cell sap were prepared
as described in Materials and Methods. The -icrosomal pellet was
resuspended in the original volume of Buffer I, 0.1 ml of each
fraction was assayed as described in Materials and Methods.

Synthetase Activity
Cell fraction nmole 32pPi incorpd into ATP/15 min


Postmitochondrial supernatant 378

Cell sap 357

Microsomal pellet 32













Table 3

Amino Acid Requirement of the P., -inosphate-ATP
Exchange Reaction


Cell sap prepared from 19-day embryos ;as divided into two
portions. One portion was dialyzed overnic-t at 4cC against Buffer I
containing 250 mM sucrose. The other portion was stored at 40C
overnight. Pyrophosphate-ATP exch-,ri,-v- was assayed with and without
added amino acid mixture as described in ts-erials and Methods.

nrole 32PPi incorpd into
ATP/15 min/mg protein

Cell sap 112

Cell sap + amino acid mixture 658

Cell sap dialyzedd) 44

Cell sap dialyzedd) + amino acid mixture 536
































































5 10


Time (min)


Figure 17.


Time course of pyrophosphate-ATP exchange assay. Cell sap was
prepared from 17-day embryonic livers and was dialyzed overnight.
The reaction conditions have been described in Materials and
Methods. ,.pr''ioximitely 5'0 ;a c-ell sap protein/ml reaction
mixture was used to determine rl'e time course. Duplicate 1 ml
reaction mixtures were assayed r: each time interval. Blanks
without added amino acid have ceen subtracted from each point.


600





500





400





300





200





100


4-,

S-
CL

C,
CO

w

E



CL
S-
o





U



0a
C~)


0
E
Cr
















300


0.2 0.4 0.6
cell sap (mg protein/ml reaction mixture)


Figure 18.


Effect of cell sap concentration on the pyrophosphate exc' i.j
reaction. Cell sap was prepared from 17-day e:,l i,oiic livers
and was dialyzed overnight. The reaction conditions have been
described in Materials and Methods. Each point represents the
average of duplicate determinations. o, incubation with added
amino acid mixture; o, incubation without added amino acid
mixture.








Figure 19 shows the variation of aminoacyl-tRNA synthetase

activity of cell saps prepared from embryos between 9 and 20 days

of development. Synthetase activity seems to be somewhat higher in

the cell sap derived from embryos at later stages of development.

However, the activity difference tlirouJhout the development stages

we studied was less than 20%.

From the results, it becomes obvious that the aminoacyl-tRNA

synthetase activity in the cell sap can not account for all the decrease

in cell sap activity in supporting protein synthesis during development.

Based on the results of this experiment and the one on the incorporation

of [ 3H] aminoacyl-tRNA, it seems reasonable to assume that the decrease

in cell sap activity is probably due to a cell sap component involved in

a stage of protein synthesis different from and possibly subsequent to

the aminoacylation of tRNA.


Ribonuclease Activity


Ribonuclease activity in various subcellular fractions of embryonic

livers at different stages of development was assayed. The results are

summarized in Table 4. Ribonuclease activity in whole liver homogenate

almost doubled between 8 and 15 days of development. The particulate

fraction contained most of the cellular ribonuclease activity. The

changes in activity of the particulate fraction with development paralleled

those of the whole cell homogenate. Before the 12th day, no ribonuclease

activity could be detected in the cell sap. However, this activity in-

creased 8-fold from day 12 to day 19. Some activity was present in

the microsomal fraction and it increased 5-fold from day 8 to day 19.

















600




500




400



300




200




100


Embryo age (days)


Figure 19.


Aminoacyl-tRNA synthetase activity of cell sap derived from
embryos of various ages. The cell saps were dialyzed over-
night and pyrophosphate-ATP exchange assayed was performed
as described in Materials and Methods. Incubation was for
15 rmin at 350C. The values represent the mean S.E. of
3 to 5 separate experiments.


I _II _la.-----..~


















C)
4-) c
C)- I r-
C O O L ,

000 O-
oE>r C C
0)Cu T 00




O E *- ( -
E 3 E l) C)











0 ro S-
C- O- N T

ro L r >v,-

O ) ) CU L*r-
) a)- -' C)

0 4- -0 -0
O- -. rC m S-










0 0 0o
EO O- ) o
C -.a -I-' N
0- C) 4-' c 3--





ro ro n
*r- S- C) C1 *r- E



: 4-' -
O ) --o Q n C
0- .- C
SC)4-' 3 C +- C)

0 S- S 4-- ) &>



0 E. L0
0)- 0 C C 1




X- U0- 0





E r>_
0L C3








0 0- .- -
O4- C *-


o0 C) CL
*- ( L

0t U)4)0 00)
N Lr -C >









3O C D.- :
0 -0) -r )
SS- o )-5 : w











>-4--) -- W C ) >.
) 0 S-
C-OOL 0 C)







C C C)O n UE
X 0 0 .
U)4- 0 1 C
0 SO ro 0-0


4- 0 C) (0 C
0)0 U)


E n i- 0 ro

0 ct U)o E) c0
&- 0*- O O ro r
0)o ~ 01r


CL
co



V)














0
0



r-
E












u





ro C)
4 r-
r-


1 '+-
0









C
0











0







0






OJ
an










E


CO
(,.--


CO Ci IDO O-)


a



o--
5-





S- 0)
rC





O
0



u
4-'











(U
0
5- e













0- 0

>U
-











C)


0 -
.0 C)

4-
5-0
c0
> w

U)

03

4- E
*O- 0
> -
*^ 4-
4-'
U

C)

0r




0
*Q
*t-
Q:








Measurement of cell sap inhibitor activity against bovine pan-

creatic ribonuclease showed that no inhibition could be.detected in

the cell sap derived from embryos before 15 days of age (Table 5).

Twenty to thirty percent inhibition was observed after day 17. Similar

levels of inhibition were seen when the inhibitor activity was assayed

against the endogeneous ribonuclease in the liver particulate fraction.

The amount of inhibition observed in the older embryos was barely within

the range of significance. Many mechanisms could be responsible for this

small decrease in ribonuclease activity. One would be the presence of a

specific inhibitor. Another possible explanation would be that such

inhibition was nonspecifically caused by the adsorption of added ribo-

nuclease to some cell sap component. The third reason may be that the

assay method is not sensitive enough to detect changes in the endo-

nuclease activity. Since the amount of substrate RNA used in the assay

was in large excess, it is less likely that the inhibition was due to

c:,i''pti tion for the substrate. The results are in line with that of

Roth (1962). He reported 30-60% inhibition for pancreatic ribonuclease

by adult chicken liver high speed supernatants under similar assay

conditions, but no inhibition by supernatant from 8-day and 9-day

whole embryos.

Increase in cellular protein synthesis has been demonstrated to

be associated with decreased ribonuclease activity and/or increased

ribonuclease inhibitor activity, and vice versa (Shortman, 1962;

Arora and de Lamirande, 1967; Moriyal;.a et a-., 1969; Sheppard et al.,

1970). It seems possible that the increase in ribonuclease activity

of cell sap with increasing developmental age may be connected with the

decrease in amino acid incorporating activity of the cell sap.












Table 5

Ribonuclease Inhibitor Activity in Cell Sap Derived
from Embryos of Various Ages


Livers were homogenized in 9 volumes of Buffer I containing 250
mM sucrose. The reaction mixture in 1 ml contained 0.0125 pjg bovine
pancreatic ribonuclease or 0.1 ml particulate fraction (resuspended
in same original volume), 0.4 ml cell sap, 0.4 ml of 1% yeast FP::
and 0.1 ml of .;1i mM Tris-HCl (pH 7.4). The mixture was incubated
at 350C for 60 rin and precipitated with 0.5 ml of 0.75% uranyl
acetate in 25% HC104 and treated as described in Materials and Methods.
Each value represents mean + S.E.


Embr'yo-, age % inhibition
(days)
Pancreatic RNase Particulate Rnase


8 2+4

12 4 +3 6+4

15 10 + 4

17 21 + 6

19 27 + 5 25 + 8


24 + 8








Distribution and Activity of Free and



Distribution of Free and Membrane-Bound Polysomes


Since serum proteins are probably synthesized by membrane-bound

polysomes and early embryonic liver cells contain very little endo-

plasmic reticulum, we looked for a correlation between the distribution

of membrane-bound and free polysomes and changes in protein synthetic

capacity during development. The amount of membrane-bound and free

polysomes was estimated by a modification of the method of Blobel and

Potter (1967). Because a significant amount of membrane-bound poly-

somes tendsto sediment in the mitochondrial fraction, total cytoplasmic

polysomes and free polysomes are separated from postnuclear supernatant.

Membrane-bound polysome content was determined as the difference between

total anJ free polysome content thus circumventing incomplete recovery

of membrane-bound polysomes from the boundary of two sucrose layers.

Usually the nuclear fraction contained 7-10% of the total cellular

RNA.

The total polysome content per gram of liver at various ages was

relatively constant (Figure 20). The free polysome content decreased

markedly between day 8 and day 18, whereas the membrane-bound polysome

content increased during the same period. Hence the percentage of total

polysomes present on membranes increased 2-fold as development progressed.

To ascertain that ribosomes did not readsorb the membrane during

isolation, a mixing experiment was performed. Postnuclear supernatant

was prepared from a 1:1 mixture of 10-day and 17-day liver homogenates.

Free and membrane-bound polysome contents were measured in this mixture


























Figure 20. Distribution of free and membrane-bound polysomes in
livers at various stages of development. Livers were
hoi..,-nized in 3 volumes of Buffer I containing 250 mM
sucrose. Free and membrane-bound polysomes were
fractionated from the postnuclear supernatant as described
in Materials and Methods. The values are the means +
S.E. of 3 to 4 separate experiments. The results were
expressed in mg RNA per ml homcn!--rite. o, total polysomes;
e, free polysomes; A, membrane-bound polysomes; 2, ,
membrane-bound in total polysomes.
















1:8

1.6


1.4


1.2

1.0

0.8


- ~ilI


Embryo age (days)


T T _

II




~2~--I







4 t I


0.6

0.4


0.2?


60 -


40


8 10 12 14 16 18


2 22 24


~,I t I I ------ L- -PI--I- -


- -


w--









as well as in the 10-day and 17-day controls. The amount of free and

membrane-bound polysomes found in the mixture was approximately the same

as the avera'j_ of the two controls (Table 6). Therefore, reassociation

of free ribosomes with the membrane during isolation was not significant.

These experiments indicate that the proportion of membrane-bound

polysomes in total polysomes increases during development. Chemical

measurements by Pollak and Ward (1967) showed that the phospholipid

content of the microsomal fraction increases during development. An

increase in rough endoplasmic reticulum seems to be a general phenomenon

in cells undergoing rapid growth such as in the neonatal (Dallner et al.,

1966) and re,.:rnrating rat liver (Tata, 1970). The exact function of

the ribosome-membrane assembly is not clear, since an accumulation of

intracellular protein also occurs during the same period. Because of

the increase in serum protein concentration in blood during development,

it seems highly probable that the increase in membrane-bound polysomes

in c.'Lb.';-nic chick liver results in the increased synthesis of secreted

proteins.


Activity of Free and Membrane-Bound Polysomes


The preceding experiments suggest that the distribution of free

and membrane-bound polysomes changes while the amino acid incorporating

activity of total polysomes remains the same during development. It

was not known whether the activity of the free and membrane-bound

pol.:comes is the same or whether their activities chan'j.: during develop-

ment. To resolve this question, the amino acid incorporating activity

of these two types of polysomes was assayed.


















L () )
S 0 :3 C
i-- ro (L)
U > -

4-- S- -C C)

O 0
Ca .-

m= 3


LO >- r--
o* C








*4 C -0
4) 4-' 0 C-






r- 0)UL
3 0 OC
CA E -0 S









*o
r I L

E X-

o-- S .-
OT) W
UECl
0 *r-' Cr-


D n (nr
N -0 r_


















*E 3 --:
C 1 0 C)




0
UC 0


) 4--0 (D










S- C ,-














E
*3 LL
C -

CAE4-'





OE *-







*r o 0)
3 X 0 0 -'

0 E +-' C











4LE O
ONL L
*i CT3 n4-






S 3 0 C
0 S.- S.









2 3A C


Ct
4-)
0


0)
cL
0

o
0





0
E
c C




I



C
0r-











C) -
m I


0 )


E

0 r
Co

So


Q 0
S.-
LL


'4-
4I-

L3



-D


CA
O


i- in0 i


E
0


0












O<-
E


LO
- E











0




























0
C-A

E
0


0
Q-








0)
E
C)
i-
Q-








E
o
CD


Sc


C-

















1u


0








For studying amino acid incorporation, nembrane-bound and free

polysomes were isolated from postmitochondrial rather than postnuclear

supernatant, because polysomes were more dec'-:-ed in the postnuclear

supernatant. The disadvantage of the procedure used is that con-

siderable losses of membrane-bound polysomes occur during separation

of postmitochondrial supernatant.

The amino acid incorporating activity of free and membrane-bound

polysomes prepared from 12-day, 15-day and 19-day embryonic livers is

shown in Table 7. Because of the small size and low membrane-bound

polysome content of liver, polysome analyses from embryos younger

than 12 days were not significant. The activity of free polysomes

derived from embryos of these three ages was approximately the same.

In contrast, the activity of membrane-bound polysomes appeared to

decrease with age. The activity of free polysomes was found to be

hinicr than that of membrane-bound polysomes derived from embryos of

the same age. The difference between the activities of the two

types of polysomes was more pronounced in the older embryos compared

with that in j.rung embryos. However, polyso-es could be released from

the 19-day membrane-bound polysome preparation by detererint treatment

(Figure 21). The greater amount of ultraviolet absorbing material in

the upper fractions of the membrane-bound versus free polysome prepa-

rations is probably membranous material released upon detergent treat-

ment. The time course of incorporation sho,.ed that these two types of

polysomes incorporated amino acid at the sar.e initial rate, however;

with membrane-bound polysomes it leveled off earlier than that with




















h- -, O
cn o1


V)


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cL
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COt
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Figure 21.


Sucrose gradient profiles of membrane-bound and free
polysomes prepared from 19-day c!i.br ,'onic livers without
using detergent. Polysomes were analyzed after addition
of de'L,-r,-:nt to the membrane-bound polysome preparation
as described in Materials and Methods. Approximately 3.0
A260 units of polysomes were layered over 36 ml 15-40D
sucrose qir.,dients centrifuged and monitored as described.
----, represents free polysomes; ---- represents
membrane-bound polysomes.




















I
I
I
0.4 -








0.3









0.2 \
I

I
















0.1 -
I



































10 20 30
ml from meniscus
ml from meniscus









free polysomes (Figure 22). Once again the difference was greater in

older embryos.

In the-above experiment, the two types of polysomes were prepared

without using detergent. Because the membrane was still associated with

polysomes during the assay of amino acid incorporation, the observed

difference in activity could not be attributed solely to the polysome.

In other words, the presence of membrane may be inhibitory to amino

acid incorporation. To test this possibility, both free and membrane-

bound polysomes were treated with Triton X-100 and reisolated. To

obtain undegraded polysomes, yeast RNA had to be added to the polysome

preparation during detergent treatment. In the absence of yeast RNA,

membrane-bound polysomes were degraded extensively as shown by the

sucrose gradient profiles in Figure 23. Unlike the finding with rat

liver (Blobel and Potter, 1966), addition of cell sap to the membrane-

bound polysomes during Triton X-100 treatment did not protect polysomes

from degradation (Figure 23B). In contrast, relatively undegraded

free polysomes were isolated in the absence as well as the presence

of yeast RNA (Figure 24). It appeared that the detergent treatment

might release or activate some ribonuclease present in the polysome

preparations. Therefore, ribonuclease activity in the polysome prep-

arations was assayed (Table 8). Before Triton treatment, the ribo-

nuclease activity in free polysomes from 18-day embryos was low (just

twice the detectable level in this assay). That in the membrane-bound

polysomes was 5-fold higher. After membrane was removed by detergent,

the polysomes originally bound to the membrane contained one third

their original ribonuclease activity. Since more ribonuclease activity























































[HE] JO uo.ieJodJou.L wnuLxeui %


-o

cx


In aI
in 0
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c- o re
ro *.C S
4- E -.

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0 C0' 'D CL 0




Ln C 4-D a



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.c E Wrn






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)-- *r-- ( E C
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ousAl [l] JO uoLqejodJo3uL wnwixewu %


OC
0

























Figure 23.


Sucrose gradient profiles of membrane-bound polysomes
after Triton X-100 treatment. Membrane-bound polysomes
prepared from 19-day embryonic livers (approximately 20
A260 units/ml) were divided into three portions. Each
portion was treated with 20% Triton X-100 to a final
concentration of 1% (A) in the presence of 20 A260 units/
ml low molecular weight yeast RNA (B) in the presence of
cell sap (approximately 3 mg protein/ml) (C) in the
absence of yeast RNA and cell sap. Each portion was
layered over 2 ml of 1.0 M sucrose in Buffer I and
centrifuged at 226,000 X gmax for 2.5 hrs to resediment
polysomes. The pellets were resuspended in Buffer I
and approximately 4.0 ,o.0 units of each sample were
layered on a 36 ml 15-40" sucrose gradient, centrifuged
and monitored as described.









0.2





0..1


0.3





0.2





0.1






0.3





0.2





0.1


20 30


ml from meniscus

























Figure 24.


Sucrose gradient profiles of free polysomes after Triton
X-l00 treatment. Free poly-om-s prepared from 19-day
eibr',,onic livers (approximately 20 A260 units/ml) were
divided into 2 portions. Each portion was treated with
20% Triton X-100 to a final concentration of 1% (A) in the
presence of 20 A260 units/il of low molecular yeast RNA
(B) in the absence of yeast RNA. Each sample was layered
over 2.0 ml of 1.0 M sucrose in Buffer I and centrifuged
at 226,000 X gmax for 2.5 hrs to resediment polysomes.
The pellets were resuspended in Buffer I and approximately
6.0 A260 units of each sample were layered on a 36 ml 15-40
sucrose gradient and centrifuged and monitored as described.














0.3





0.2


10 20 30

ml from meniscus













Table 8

Ribonuclease Activity in Free and Membrane-Bound Polysomes


Free and membrane-bound polysomes were prepared as described in
Materials and Methods. Membrane-bound polysomes were treated with
20 Triton X-100 to a final concentration of 1%. The mixture was
layered over 2.0 ml of 1.0 M sucrose in Buffer I and centrifuged at
226,000 X gmax for 2.5 hours to sediment polysomes. The super-
natant was diluted 4 times and centrifuged at 226,000 X gmax for 1
hour. The pellet thus obtained was resuspended in Buffer I of the
same volume as the original membrane-bound polysomes preparation and
designated "'r.j:eibranous material." Ribonuclease activity was assayed
as described in Materials and Methods. Incubation was at 350C for
60 min.

12-day 18-day
embryo embryo

AA260!/i rRNA

Before Triton X-100 treatment

Free polysomes 0.043 0.061

Membrane-bound polysomes 0.144 0.306

After Triton X-100 treatment

Membrane-bound polysome 0.072 0.093


Membranous material


__


0.401








was found in the membranous material than was present with the membrane--

bound pol',omes, the detui_ ,r-:t appears to enhance the nuclease activity.

Similar results were obtained for polysomes derived from 12-day embryos

but the nuclease activity was much lower in all fractions as compared

with 18-day embryos. The results suggest that the ribonuclease

activity found in polysome preparations is associated with the membrane

rather than with the polysome. The small degree of deiradation of

free polysomes upon detergent treatment shcwn in Figure 24 may be

due to some membrane contamination in the free polysome preparations.

The A 260/At ratios of the Triton X-100 treated free and membrane-

bound polysomes were 1.75-1.88 which indicated that the polysomes were

almost completely devoid of membrane. The amino acid incorporating

activity of the detergent treated polysomes is shown in Table 7. No

difference in activity was observed between free and membrane-bound

polysomes derived from embryos of the ages tested. This result suggests

that the activity difference observed between free and membrane-bound

polysomes before deterrent treatment is probably due to some component

present in the membrane. Ribonuclease activity found in the membrane may

be that one component. The more rapid decay of amino acid incorporating

activity of membrane-bound polysomes as cc-pared with that of free

polysomes is consistent with a membrane-bound nuclease active during

incubation for amino acid incorporation. Ti.- hi:i-r' nuclease activity

in the membrane-bound polysomes of older erryos could also explain the

relative activity difference between membrare-bound and free polysomes.

It is interesting to note that deter-,'nt treatment has been shown to

alter the behavior of membrane-bound polysces by other workers









(Bloemendal et al., 1967; McDonald and Konner, 1971). However, the

difference may also be ascribed to a different average size of the

rL.;NA molecules in the two polysomes.

The relative amino acid incorporating activity of membrane-bound

and free polysomes seems to depend upon the method as well as the ionic

conditions used for isolation of the two polysome fractions, since in

rat liver cells, membrane-bound polysomes have been reported to be

more (Tata and Williams-Ashman, 1967; Pain et al., 1974), equally

(Bloemendal et al., 1967; Takagi and Ogata, 1968), or less (Rayr'tti,

Lawford and Campbell, 1969; McDonald and Konner, 1971) active than free

polysomes, apparently the result of different isolation procedures.

Under the assay conditions described above, we detected no difference

between the activity of free and membrane-bound polysomes. However,

membrane seems to affect amino acid incorporation by polysomes in the

cell-free system.


Albumin Synthesis


As a first step in our study of the molecular events that

initiate and control albumin synthesis during development, it was

decided to measure albumin synthesis in the cell-free system. Free and

membrane-bound polysomes were incubated for amino acid incorporation.

After incubation, the reaction mixtures were centrifuged to sediment

polysomes.. Approximately 30-40% of the hot trichloroacetic insoluble

counts was released into the supernatant from the free polysomes and

i5-20" of counts was released from the membrane-bound polysomes. The

supernatants from each reaction mixture were dialyzed extensively and








then subjected to gel electrophoresis at pH 8.9 in a 7.5% polyacrylamide

gel. Under these conditions, authentic chicken serum albumin migrates

faster than most other serum proteins so that it appears as a discrete

band behind the bromphenol blue tracking dye. Figure 25 shows the gel

patterns of the dialyzed supernatants derived from 16-day embryos.

No significant radioactive peak appeared to migrate at the position

of standard albumin. Finding no albumin in the supernatant, the

polysomal pellets were resuspended in buffer, frozen, thawed, sonicated

and centrifuged again. About 25-30% of the hot trichloroacetic acid

insoluble radioactivity was released into the supernatant by such

treatment. The released material was dialyzed and analyzed by gel

electrophoresis. Figure 26 shows the gel pattern of the materials

released from the free and membrane-bound polysomes derived from 16-day

embryos. In the free polysome preparation, some radioactivity was

present in the region corresponding to albumin standard, but no

discrete band was observed. One major peak of radioactivity from

the membrane-bound polysome pr cFlration comigrated with standard albumin.

To characterize this peak further, carrier chicken serum albumin was

added to another aliquot of the sample and the mixture was reacted

with ,o.it antiserum against chicken serum albumin. After immuno-

precipitation, the supernatant was subjected to gel electrophoresis.

The antiserum removed the peak of radioactivity which had comigrated

with albumin (Figure 27). The ii;1munoprecipitate was washed several times,

dissolved, denatured with SDS and subjected to SDS polyacrylamide gel

electrophoresis (Figure 28). A radioactive band was observed comigrating

with standard albumin run on a sister gel. The radioactivity in the band






















10



8



6



4




2







Figure 25.


---- ---- ----- --
5 10 15
Fraction No.


Polyacrylamide gel electrophoresis of cell-free protein products
released from membrane-bound and free polysomes before sonication.
Membrane-bound and free polys2-es prepared from 16-day embryonic
livers were incubated for protein synthesis as described in
Materials and Methods. After incubation, the reaction mixture
was centrif..E-.1.-: to sediment polysomes and the supernatant was
dialyzed extensively and analyzed by gel electrophoresis as
described in Materials and M:etods. Approximately 9000 cpm and
5C0,l1 cpm of trichloroacetic acid insoluble material from free and
membrane-bound polysomes respectively were applied to the gels.
The protein staining band of standard albumin is indicated in the
graph. o, free polysomes, g, -e~brare-bound polysomes.

























Figure 26.


Polyacrylamide gel electrophoresis of cell-free reaction
products released from membrane-bound and free polysomes
by sonication. Conditions for amino acid incorporation
and analysis of the products have been described in
Materials and Methods. About 7,000 to 15,000 cpm of
trichloroacetic acid insoluble material was applied to
the gels. The protein staining band of standard albumin
is indicated in the graph. A) 16-day membrane-bound
polysomes (B) 16-day free polysomes (C) 12-day membrane-
boirid polysomes (D) 19-day mer;Drane-bound polysomes.


















I
CNj
O

E




10 20 30
Fraction No.


12

10

8

6
4

2


12


10 20 30
Fraction No.


Fraction No.


10 20 30
Fraction No.


------L------i------I I__























I

I




I


1






I
O
1I




I
t


20 25 30
^20 25 30


Fraction No.


Figure 27.


Polyacrylamide gel electrophoresis of cell-free protein products
of 16-day membrane-bound poiysomes remaining after precipitation
with antiserum against albumin. The cell-free protein products
were incubated with antiserum against alb-umin. After removal of
the immunoprecipitate by centrifi.',tion, the supernatant was
subjected to gel electrophoresis as described in Materials and
Methods. The dotted line is the profile of the radioactive
products present prior to immunoprecipitation. The protein
staining band of standard serum albumin is indicated in the
grc, ph.


It
'i

It


it

























8





6


',j

CL

0
4
E


5 10 15
5 10 15


Fraction No.


Figure 28.


SDS-polyacrylamide gel electrophoresis of the cell-free protein
products precipitated with antiserum against albumin. The cell-
free products from 16-day membrane-bound polysomes were incubated
with antiserum against albumin. The immunoprecipitate was washed
and dissolved in 20 mM Tris-HCl (pH 7.4), 1% SDS and 1% B-
mercaptoethanol. The c.3ple was subjected to SDS-polyacrylamide
gel electrophoresis as described in Materials and Methods. The
protein staining band of standard serum albumin is indicated in
the graph.


____ i








r,,-cr'e its more than 70% of total radioactivity recovered from the

gel. There was some radioactivity present in a lower molecular weight

region which'may represent either incomplete albumin chains or non-

specific precipitation. These experiments indicate that the major peak

of radioactivity represents newly synthesized albumin. The gel patterns

of products synthesized by membrane-bound polysomes derived from 12-day

and 19-day embryos are shown in Figure 26. They are very similar to the

16-day pattern.

To determine the amount of albumin synthesized, each sample

was reacted with antiserum against albumin and the resulting immuno-

precipitate was counted. Table 9 summarizes the results. The per-

centages of albumin in total polypeptides synthesized by membrane-

bound polysomes were approximately the same at different ages. The

amount of albumin synthesized by free polysomes was much less than

that synthesized by membrane-bound polysomes. The result suilests

that membrane-bound polysomes are the major site for albumin synthesis

in embryonic chick liver cells. It is interesting that this phenomenon

is seen even in the 12-day cribryonic livers which contain relatively

fewer membrane-bound polysomes. The 8- to 10-fold difference in albumin

synthesis by membrane-bound versus free polysomes is in the same range

as the results of similar investigations in mammalian systems (Shafritz,

1974b; Koga and Tamaoki, 1974).

Although the amount of albumin synthesized per weight of membrane-

bound polysomes remained constant during development, the possibility

remains that the increase in albumin synthesis in vivo is due to the

increase in the content of membrane-bound polysomes during development.




87










r-

nO
4->*r-



ON
(0 0 L




-p.
*r- 4-


0> .







F-r-
0-
*0 S-


















4_ --, 4 ,- -





Q) 0 0- E m m CO
a 0 oa E (D ,r- --

.0 0 .

S- rO- -

u i-
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Ot OC)


E m 0












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0-







0 r- --
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r- O O O O
-E 1 CO ro

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*- *o m i &
-la u4- *r +' co j- r-. n






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A cell-free system composed of low speed supernatant of liver homogenate

seemed to be suitable for testing this possibility for the following

reasons. The low speed supernatant contains both membrane-bound and

free polysomes present in their in vivo ratio. This system has been

shown to synthesize pol.,'pptides with an in vivo-like pattern (Hill,

Wilson and Hoagland, 1972). Another advantage of this system is that

it requires only small liver samples which allows the study of early

embryos.

In the following experiments, the cell-free reaction products

synthesized by 3,000 X g supernatant derived from 9-day, 13-day and

16-day er-,.r,.'.s were compared. After incubation for protein synthesis,

the reaction mixtures were centrifuged to sediment microsomes. The

supernatants of 9-day, 13-day and 16-day reaction mixtures so prepared

contained 35%, 2- and 17% of the total hot trichloroacetic acid in-

soluble counts respectively. This is probably due to the presence of

different amounts of membrane-bound polysomes in the samples, because

a lower percentage of radioactivity is released from membrane-bound

polysomes as shown in the previous experiment and in the results of

other workers (Andrews and Tata, 1971). The supernatants did not

contain albumin as judged by gel electrophoresis and immunoprecipitation

(not shown). The microsomal pellets were frozen, thawed and sonicated.

The sonic supernatants were dialyzed and analyzed by gel electrophoresis.

The gel patterns are shown in Figure 29. In each sample, a radioactive

peak comigrated with standard albumin. The amount of albumin synthesized

in each sample was again estimated by counting the immunoprecipate after

reaction with anitserum against albumin. The relative proportion of

albumin synthesized by 9-day ci'. :-. was much less than that synthesized



























Figure 29. Polyacrylamide gel electrophoresis of cell-free protein
products synthesized by 3,000 X g supernatant from livers
at various stages of development. Conditions for amino
acid incorporation and analysis of products have been
described in Materials and Methods. About 7,G'"0 to
9,000 cpm of trichloroacetic acid insoluble material
was applied to each gel. The protein staining band of
standard albumin is indicated in the graph. (A) 9-day
embryos (B) 13-day embryos (C) 16-day embryos.










(A)
6
I



C3






10 20 30
Fraction No.

6 (B)












Fraction No.
6- (c)
C\.J
cV 5














E 2 1i ",
3t


E 3 t I \ ----9a-r--- ]\ \


10 20 30
Fraction No.
6 (C)
5










10 20 3 0
Fraction No.




91



by 13-day and 16-day embryos (Table 10). The increase in the amount

of albumin synthesis with developmental age agrees with the increase

in albumin cr.~.irctration in the serum. This result confirms the

previously postulated correlation between an increase in the proportion

of membrane-bound polysomes and an increase in specific protein synthesis.





92










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